【摘要】 目的:在大肠杆菌中重组表达贝类主要过敏原—原肌球蛋白(Pen a 1),并且评估其IgE结合活性。方法:首先基于Pen a 1的已知序列人工合成Pen a 1基因,其次将其与质粒p ET-28a连接以构建重组表达质粒pET-28a(+)-Pen a 1,经测序、酶切鉴定后导入表达宿主E. coli BL21(DE3)细胞中。通过IPTG诱导,重组表达Pen a 1蛋白,镍柱纯化表达产物,并采用SDS-PAGE电泳及质谱技术鉴定所得重组蛋白。最后应用贝类过敏患者采用LICA技术检测该重组蛋白的IgE结合活性。结果:PCR结果表明,目的基因全长为930 bp,与理论预测值一致。SDS-PAGE电泳结果显示,目的蛋白在宿主菌内以可溶性蛋白的形式高效表达,分子量约为36 k D,大小与理论值相符。质谱鉴定结果进一步说明所得的重组蛋白为原肌球蛋白Pen a 1。免疫分析结果表明该蛋白具有较高的IgE结合能力。结论:成功表达、纯化出有良好免疫学活性的重组原肌球蛋白Pen a 1,为进一步研究Pen a 1在贝类动物如虾类过敏的诊断和治疗中作用奠定基础。更多还原

【Abstract】 Objective: To clone and express Pen a 1 as a major allergen from Penaeus aztecus in E.coli, and to analyze its immunoglobulin E(IgE)binding ability. Methods: The gene coding Pen a 1 protein sequences were synthesized and cloned into the Bam HI and Hind III sites of p ET28 a(+) expression vector. After sequencing and identified by restriction endonuclease digestion, the verified p ET-28 a(+)-Pen a1 was transformed into E. coli BL21 cells. The recombinant expression of Pen a 1 was induced by IPTG. The recombinant protein was purified using Ni+-NTA affinity column chromatography, characterized by sodium dodecyl sulphate SDS-PAGE and MS, and tested by LICA for IgE reactivity with sera from individuals with shellfish allergy. Results: PCR analysis showed that the size of the target gene fragment was about 930 bp, which was consistent with the theoretical predictions. SDS-PAGE showed that the recombinant protein was highly expressed in the form of soluble protein in E. coli, and its molecular weight was about 36 kD, which was in accordance with the theoretical value. The results of mass spectrometry further revealed that the expression product was tropomyosin Pen a 1. Immunological analysis indicated the protein had high IgE binding ability. Conclusion: Recombinant tropomyosin Pen a 1 with good immunogenicity was successfully produced, which laid the foundation for further study on the role of Pen a 1 in the diagnosis and treatment of allergies including shrimp allergy.更多还原